Sequences containing the vpu gene and vectors therefore methods of preparation and use

ABSTRACT

DNA segments encoding the vpu gene and a vector encoding the vpu gene are disclosed. These sequences containing the vpu gene can be used to express a protein that has antigenic determinants that can be used to screen for people having the HIV-1 virus.

This is a divisional of copending application Ser. No. 07/193,321 nowU.S. Pat. No. 5,043,262 filed on May 12, 1988.

The present invention is directed to a new purified polypeptide, amethod of producing this polypeptide, an antibody to this protein, anassay for detecting in biological specimens the presence of an antibodyto the antigenic determinants present in said polypeptide, and an assayfor the negative effect of this protein upon viral replication.

The human immunodeficiency virus (HIV-I, also referred to as HTLV-III,LAV or HTLV-III/LAV) is the etiological agent of the acquired immunedeficiency syndrome (AIDS) and related disorders. [Barre-Sinoussi, etal., Science 220: 868-871 (1983); Gallo et al, Science 224: 500-503(1984); Lane et al, Levy et al, Science 225: 840-842 (1984); Popovic etal, Science 224: 497-500 (1984); Sarngadharan et al, Science 224:506-508 (1984); Siegal et al, N. Engl. J. Med. 305: 1439-1444 (1981)].This disease is characterized by a long asymptomatic period followed bythe progressive degeneration of the immune system and the centralnervous system. Studies of the virus indicate that replication is highlyregulated, and both latent and lytic infection of the CD4 positivehelper subset of T-lymphocytes occur in tissue culture. [Zagury et al,Science 231: 850-853 (1986)]. The expression of the virus in infectedpatients also appears to be regulated as the titer of infectious virusremains low throughout the course of the disease. Molecular studies ofthe replication and genomic organization of HIV-I show that it encodesat least eight genes [Ratner et al, Nature 313: 277-284 (1985);Sanchez-Pescador et al, Science 227: 484-492 (1985); Muesing et al,Nature 313: 450-457 (1985); Wain-Hobson et al, Cell 40: 9-17 (1985)].Three of the genes, the gag, pol and env genes are common to allretroviruses. However, the genome also encodes five additional genesthat are not common to most retrovirus, the sor, tat, art and 3'orf andR genes [Sodroski et al, Science 231: 1549-1553 (1986); Arya et al,Science 229: 69-73 (1985); Sodroski et al, Science 227: 171-173 (1985);Sodroski et al, Nature 321: 412-417 (1986); Feinberg et al, Cell 46:807-817 (1986); Wong-Staal et al, AIDS Res. and Human Retroviruses 3:33-39 (1987), which are all incorporated herein by reference.]

Nucleotide sequences from viral genomes of other retroviruses namelyHIV-II and Simian immunodeficiency virus (SIV) (previously referred toas STLV-III) also contain tat and art regulatory sequences and showtransactivation in addition to containing the structural genes. [Guyaderet al, Nature 326: 662-669 (1987); Chakrabarti et al, Nature 328:543-547 (1987)]. It would be useful to have a method for readilydistinguishing between HIV-I and other retroviruses, such as HIV-II.

One of the problems with developing a vaccine to the AIDS virus is thatthe various strains of HIV are not highly conserved. And, the envelopeprotein, which has been used as a target because it goes to the surfaceof the cell, shows a great deal of variability between strains.Accordingly, it would be useful if there was another protein that wentto the surface of the cell and was highly conserved in different HIVstrains.

Any HIV product which demonstrates a negative effect upon virusreplication requires evaluation for possible therapeutic applications.Specifically, an assay for the negative effect is required so that drugscan be evaluated for possible usefulness as agents to enhance or mimicthe negative effect.

SUMMARY OF INVENTION

We have now discovered a protein expressed by cells infected with HIV-I.The protein has a molecular weight of approximately 16 kD, but iscleaved to a 15 kD form. This protein has antigenic determinants andpatient antisera from AIDS infected patients recognize both forms ofthis protein whereas the antisera of normal patients do not recognizeit.

This protein appears to have a negative effect upon the replication ofthe HIV-1 virus in cultured human CD4+ lymphocytes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the genetic organization of the central region of the Elistrain of HIV-1 compared to SIV and the ROD strain of HIV-2.

FIG. 1B shows the alignment of the vpu gene protein sequence.

FIG. 1C is a hydrophobic profile of the predicted vpu gene product.

FIG. 2A-D are radiograms showing the in vitro characterization of thevpu gene product.

FIG. 3A-D are radiograms showing the identification of the vpu geneproduct in cell lines expressing constitutively proteins encoded by the3' half of the Eli strain of HIV-1.

FIG. 4 is a schematic showing the plasmid pEU used to synthesizemessenger RNA.

FIG. 5A is a schematic showing the recombinant provirus constructed forcomparison of viruses capable of producing the vpu gene product withviruses incapable of making it.

FIG. 5B is an autoradiogram showing the differences in production ofviral products resulting following transfection of lymphocytes withproviral plasmids either capable or incapable of producing the vpu geneproduct.

DESCRIPTION OF INVENTION

We have now discovered that HIV-1 encodes an additional protein and thatantibodies to this protein are detected in the sera of people infectedwith HIV-1. Further, this protein can be used to distinguish HIV-1isolates from the other human and simian immunodeficiency viruses (HIV-2ans SIV) [Guyader, M., et al, Nature 326, supra (1987)]; Hirsch, V., etal, Cell 49: 307-319 (1987); Chakrabarti, L, et al, Nature 328, supra]since we have also discovered that the latter viruses do not encode asimilar protein.

This protein is expressed by a DNA segment containing a sufficientnumber of nucleotides corresponding to nucleotides 5541-8017 of theHIV-1 genome, where an AUG codon is inserted immediately upstream and inthe proper reading frame with this region to express a polypeptide. Thisregion typically corresponds to the U open reading frame. A schematicdiagram of this open reading frame from the region between the firstcoding exons of the tat and art genes of HIV-1 and the envelopeglycoprotein gene is pictured in FIG. 1A. However, many strains do notcontain an AUG codon in the proper reading frame. The Eli strain ofHIV-1 does contain such an arrangement. FIG. 1A shows the geneticorganization of the central region of HIV-1 (Eli isolate, 24) comparedto SIV (20,21) and HIV-2 (ROD isolate, 19). Arrows indicate theinitiator AUG codons in the viral genes. Many HIV-1 strains have thecapacity to encode this protein which is about 80 to 82 amino acids longand synthesized from this region when initiated with an AUG codon. FIG.1B illustrates alignment of this protein sequence among various strains.The Eli strain is taken as reference. Gaps (--) were introduced usingstandard methodology to optimize the alignment. Asterisks indicate aminoacid identity. The HIV isolates compared include Eli, Mal [Alizon, M.,et al, Cell 46: 63-74 (1986)], HXBc2, BH-10, BH-8, pHXB3 [Ratner, L., etal, Nature 313: 277-283 (1985)], BRU [Wain-Hobson, S., et al, Cell 40:9-17 (1985)] and USF2 [Sanchez-Pescador, R., et al, Science 227: 484-492(1985)]. [All of the above-mentioned references are incorporated hereinby reference]. These strains are generally available. USF2 contains atermination codon at position 39, however, a-1 point frameshift resultsin a protein 43 amino acids longer that is well conserved when comparedwith the Eli reference sequence. This new protein contains a hydrophobicleader sequence (FIG. 1C), which resembles membrane transport sequences.

This suggests that the protein is transported across the cell membrane.Further, the position of the U reading frame within the viral genomesuggests that the protein forms an alternate leader sequence for theenvelope glycoprotein. Occurence of either a splicing event or aframeshift in translation of the U protein at position X would result inthe production of a fusion protein with the envelope glycoprotein. Insuch a case, the U protein would become attached to the amino terminalof the envelope glycoprotein forming a new amino terminal.

The protein, which has a molecular weight of about 16 kilodaltons (kD)can be cleaved to yield a shorter form of the protein, which has amolecular weight of about 15 kD.

Because the gene for this protein is located in an open reading framethat has been designated U [Wain-Hobson, S. et al, Cell 40:9-17 (1985)]we propose calling the viral protein U and the gene vpu for viralprotein U.

In order to more fully examine the properties of this protein, twooligopeptides were made corresponding in sequence to the hydrophilicregions of the protein based upon amino acid sequence composition. Thefirst oligopeptide designated peptide 1, corresponded to amino acids29-41, while the second oligopeptide designated peptide 2, correspondedto amino acids 73-81 (FIGS. 1B, 1C). These peptides were conjugated tokeyhole limpet hemocyanin using standard techniques and were used toraise antibody in a number, for example, 3 rabbits. After multipleinjections of the antigen, the rabbits did produce antibodies thatrecognize the immunizing oligopeptide.

FIG. 2 shows the in vitro characterization of the vpu gene product.Both, the 15 kD and 16 kD viral proteins were precipitated by the rabbitantiserum to peptide 2 (see FIG. 2A, lane 4). The antisera to peptide 1also precipitated these proteins, although more weakly. In contrast,these proteins were not precipitated by the sera from preimmune rabbitsera (FIG. 2A, lane 3). The data of FIG. 2D demonstrates that HIVseropositive patient antiserum also recognize both the 15 kD and 16 kDproteins (lane 2). This ability of the patient antisera to precipitatethe two proteins is partially competed by peptide 2 (See lane 3). All ofthe 19 sera of HIV-1 infected patients that immunoprecipitated thetruncated envelope product were also found to precipitate both the 15 kDand 16 kD proteins. These proteins were not recognized by normal patientantiserum (lane 1).

This protein appears to be highly conserved among HIV-1 proviralstrains. Indeed, all HIV-1 proviral strains isolated contain an openreading frame in the region corresponding to vpu. However, manyindividual proviral strains explored in vitro appear unable to produce aprotein from this region because of a single point mutation that preventvpu expression. Indeed, different proviral strains isolated from thesame virus isolate are heterogeneous with respect to the ability toencode vpu. For example, independent proviral clones of the IIIB isolateHXBc2, BH10, BH-8 and BH-3 differ in this regard (See FIG. 1B). Asimilar variation in the ability of individual proviral clones to encodeother viral protein, especially the 3' orf product, has also been noted.For example, in the case of the isolate of IIIB with respect to 3' orf,a mutation that truncates the protein product yields a virus thatreplicates more rapidly in culture than does the wild type [Terwilliger,E. et al, J. Virol. 60:754-760 (1986)]. Proviruses incapable ofexpressing vpu can replicate, as is demonstrated by the ability of thevirus produced by transfection with, for example, the provirus HXBc2 togrow in T-cells in culture, although this is a vpu-deficient viruses.This, however, does not rule out the possibility that the vpu productplays an important role in regulation of viral replication orpathogenesis.

Indeed, a computer assisted search for proteins similar to p15^(vpu) andp16^(vpu) found that HIV-2 and SIV do not encode a similar protein.Neither strain contained an open reading frame comparable to the Ureading frame. Both HIV-2 and SIV strains contain an open reading framemissing from that of HIV-1 isolates, the X open reading frame [Guyader,M. et al, Nature 326, supra (1987)] but there is no detectablesimilarity in the vpu protein and any protein that could be formed fromthe X open reading frame. Furthermore, none of the sera ofHIV-2-infected patients surveyed contained antibodies to the vpu productnor were antibodies to the vpu product detected in Rhesus macaquesinfected with SIV.

Thus, antisera to p15^(vpu) and p16^(vpu) can be used to distinguishamong HIV-1 and SIV or HIV-2 infections.

Additionally, the titer of patient antibodies to the vpu product canalso be used to determine the stage of HIV-1 induced disease. Byexamining the viral messenger RNAs that are used to encode regulatoryproteins [Meusing, M. A., et al, Nature 313:450-458 (1985), Arya, S. K.,et al, Science 229:69-73 (1985) and Sodroski, J. et al, Nature321:413-417 (1986)] it is apparent that vpu is removed by splicing fromsuch viral mRNAs. Accordingly, vpu should not be made in the absence ofthe art gene product as only fully spliced mRNAs accumulate in theabsence of this product. [Sodroski, Nature 321, supra; Feinberg, M. B.,et al, Cell 46, supra (1986)]. Consequently, the vpu protein is, likeother virion proteins, synthesized late in infection, and the titer ofpatient antibodies can be used to determine the stage of the disease.

Expression of the U protein can readily be carried out by the person ofordinary skill in the art by using standard techniques based upon thepresent disclosure. For example, one can prepare a DNA segmentcontaining the vpu gene, a nucleotide sequence corresponding to asufficient number of nucleotides from nucleotides 5541-8017, where anAUG codon is inserted immediately upstream and in proper reading framewith this region to express the U protein. Typically, this DNA segmentwill be inserted into a vector which contains a promoter, preferably aviral promoter, upstream of the segment. The vector preferably alsocontains an enhancer and polyadenylation signal. Preferably, one woulduse the nucleotide sequence corresponding to the U open reading frame inthe HIV provirus, however, when using a strain that does not contain anAUG initiation codon such as HXBc2, care must be taken to insure thatthe initiator sequence AUG is added in the proper reading frame such asoccurs in HIV strain Eli. Accordingly, the use of a sequencecorresponding to the U open reading frame is most preferable.

If one was to use a strain corresponding to the U open reading framefrom for example, strain HXBc2, one would have to insert an AUG codonimmediately upstream and in proper reading frame with the U open readingframe at a nucleotide corresponding to immediately before nucleotidesequence 5541 or create a point mutation to generate such a sequence.However, this can be done by standard techniques well known in the art.

By inserting a vpu gene in a standard expression vector, for example,the SP6 plasmid, using techniques well known in the art. (See FIG. 4showing creation of plasmid pEU), this plasmid can be used to synthesizethe p15^(vpu) and p16^(vpu) by standard techniques.

This protein can be used in preparing an antigen to create a vaccine,for example, a live attenuated vaccine. By using this protein it ispossible to generate an antigenic response to the protein and because ofthe aforesaid properties that the protein is found on the surface ofcells infected by the virus and because this protein appears to behighly conserved among various HIV-1 strains, the vaccine created shouldbe particularly useful.

As mentioned above, because it contains antigenic determinants, whichare highly conserved, and appear to be specific to cells infected withHIV-1 this protein is particularly useful as a diagnostic tool forassaying biological specimens to determine whether they contain cellswhich have been infected with HIV-1. These assays can be prepared usingstandard techniques. For example, one can take a predetermined sample,i.e., the biological specimen to be tested, and add an anti-idiotypicantibody or immunologically similar to the antigenic sites of thep15^(vpu) and p16^(vpu). For example, peptide 2 described herein,corresponding to amino acids 73-81 (FIG. 1B, 1C), reflects such anantigenic site. One can preferably use monoclonal anti-idiotypicantibodies. This sample is then screened to determine if there is areaction, i.e. if a complex is formed between antibody and antigen.Alternatively, one can assay with antibodies either monoclonal orpolyvalent to the antigenic determinants of the viral protein itselfusing known immunoassay methods, for example, using competitiveimmunoassays or immunometric (sandwich) assays.

Because this protein appears to have an attenuating effect upon the rateof spread of an HIV-1 infection in culture, it may form the foundationfor a screening program of drugs or other agents designed to mimic orenhance this phenomenon. For example, one can add the protein to aculture infected with an HIV-1 strain that does not express the protein,measure the degree of attenuation of replication, then screen for drugsthat enhance this attenuation effect by standard techniques. The proteincan be added by any technique well known in the art includingtransfecting the cells with an expression vector containing the vpugene. Accordingly, drugs that on their own are not clinically effectiveagainst replication of the virus could be useful in combination with theU protein to enhance the attenuation effect.

The present invention is further illustrated by the following examples.These examples are provided to aid in the understanding of the inventionand are not to be construed as a limitation thereof.

EXAMPLES

The ability of the vpu gene, i.e. the region between the first codingexon of tat and the env gene nucleotide sequences corresponding to asufficient number of nucleotides from 5541-8017 of the HIV genomecontain an AUG start codon, to encode a protein was examined byprogramming an in vitro reticulocyte translation lysate [Pelham, H.P.B.,et al, Eur. J. Biochem. 67:247-256 (1986)] with RNA synthesized in vitrousing the method of Melton, D. A., et al, Nucl. Acid. Res. 12:7035-7056(1984). RNA was made from a restriction fragment 2231 nucleotides longof an HIV provirus that spanned the region between the first codingexons of the tat, art and part of the env genes. The template for theexperiment was derived from a fragment of the provirus of the ELI strainof HIV-1 [Alizon, M. et al, Cell 46, supra ] placed 3' to the SP6bacteriophage RNA polymerase promoter [Melton, D. A., et al, supra].FIG. 4 shows the SP6 plasmid used to synthesize mRNA. A BamHl to Bgl IIfragment 2231 nucleotides long from the HIV Eli provirus that spannedthe region between the first coding exons of the tat, art and part ofthe env gene was cloned 3' to the SP6 bacteriophage RNA polymerasepromoter. Internal restriction sites used to linearize the plasmids areindicated. This strain contains an open reading frame in this regionthat initiates with an AUG codon (FIG. 1B) [Alizon, M., et al, Cell 46,supra]. The viral sequences present in this RNA transcript, as shown inFIG. 4, extend from the 5' end of the first coding exon of the tat (BamHl site) to 1839 nucleotides (Bgl II site) within the env. Theinitiation codon for the tat gene is not intact in this RNA as therestriction enzyme used, Bam Hl, cleaves the ELI proviral strain betweenthe T and the G of the tat initiation codon.

Proteins produced in the in vitro lysate using the RNA derived from thisproviral fragment were labeled with ³⁵ S-methionine and separated bysize using sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE). See FIG. 2A. pEU plasmid was linearized bydigestion at an EcoRI site located in the polylinker 3' to the HIV_(Eli)insert and used as a template for in vitro transcription by SP6 RNApolymerase as described [Pelletier, J. & Sonenberg, N., Cell 40: 515-526(1985)] except that the concentration of GTP and Cap analogue m⁷ GpppGwere raised to 0.2 and 1.0 mM respectively. Messenger RNAs were labeledwith [5-³ H] CTP and purified as described (27). In vitro translation ofequimolar amounts of RNA (equal amounts of radioactivity) was performedin reticulocyte lysate [Pelham, H.P.B., et al, Eur. J. Biochem., 67:247-256 (1986)]. Incubation was done at 30° C. for 30 minutes in thepresence of ³⁵ S-Methione. Labeled products were analyzed directly by15% SDS-PAGE (lane 2) or immunoprecipitated [Lee, T. H. et al, Cell 44:941-947] beforehand with preimmune rabbit serum (lane 3); anti-peptide 2serum (lane 4); anti-peptide 2 serum in the presence of 500 mM ofpeptide 2 (lane 5), peptide 1 (lane 6), or an unrelated peptideQEEAETATKTSSC (lane 7). Lane 1 represents a total translation reactionwith no mRNA added. The proteins synthesized in this system aredisplayed in Lane 2. The proteins precipitated by rabbit anti-peptide 2serum are also shown. Two proteins of molecular weight of approximately15 kD and 16 kD are evident in the unfractionated extract and areprecipitated by the rabbit antisera. The 15 kD and 16 kD proteins arenot precipitated by the sera from preimmune rabbit sera (lane 3). Allthree of the antisera to peptide 2 recognize both proteins (lane 4) asdo the antisera to peptide 1 albeit more weakly (data not shown). Thedata of FIG. 2A also show that peptide 2 competes for recognition of the15 kD and 16 kD proteins by antisera (lane 5). However, peptide 1 (lane6) or an unrelated peptide do not compare with anit-peptide 2 serum(lane 7).

RNA from other proviral fragments was prepared for analysis of the invitro translation products. In one set of experiments, the template usedfor synthesis of RNA was truncated by restriction enzyme cleavage either7 nucleotides 5' to the proposed AUG codon (Rsa I stie) or 30nucleotides 3' to the proposed AUG codon (Bbv I site) (see FIG. 4). Nospecific protein products recognized by anti-peptide 2 antiserum wereobserved in these experiments (FIG. 2b, lane 1 and 2). When the templateused for synthesis of RNA was cleaved 102 nucleotides 3' to the proposedstop codon (NdeI site), the 15 kD and 16 kD proteins were detected usinganti-peptide 2 serum (FIG. 2B) (lane 3 and 4). pEU plasmid waslinearized with the following restriction enzymes RsaI (lane 1); Bbv I(lane 2) and Nde I (lanes 3 and 4). SP6 generated RNAs were translatedin vitro and immunoprecipitation was performed on the labeled productsusing anti-peptide 2 serum (lanes 1, 2 and 4). Lane 3 represents a totalin vitro translation reaction.

The sequence of the HXBc2 clone of the IIIB isolate, as aforesaid,suggests that the RNA derived from this region should be incapable ofproducing the proteins as it lacks an initiation codon at the 5' end ofthe open reading frame. RNA was prepared by in vitro transcription ofthe fragment of the HXBc2 proviral clone that spans the region betweenthe first coding exons of tat and art and part of the env gene (Bam H1site; nucleotide 8017) and corresponds to the Eli fragment present inEu. This RNA was used to program a reticulocyte lysate in a similarmanner as described above. As was the case for the ELI proviral RNA, theinitiation codon of the tat was deleted from the RNA. Proteinscorresponding to the 15 kD and 16 kD products were not found in eitherthe total ³⁵ S-methionine-labelled extract or in the precipitatesobtained using anti-peptide 2 serum (FIG. 2C, lane 1, 2). A plasmid(pXU) containing a Sal I (position 5441) to BamH1 (position 8017)restriction fragment from the HXBc2 Dtat-1 proviral DNA clone [Dayton,A.I., Cell 44: 941-947] located 3' to the SP6 RNA polymerase promoter[Melton, D.A., et al, Nucl. Acid. Res. 12: 7035-7056 (1984)] wasconstructed by standard techniques. pXU was linearized with EcoR1 andused as template for in vitro transcription. After in vitro translation,the labelled products were analyzed directly on 15% SDS PAGE (lane 1) orimmunoprecipitated beforehand with anti-peptide 2 serum (lane 2) or anHIV-1 infected patient serum (lane 3). However, immunoprecipitation withHIV-1 infected patient serum clearly showed the presence of truncatedenvelope product (lane 3).

FIG. 2D shows the cross-reactivity between this protein and the HIV-2and SIV viruses. After in vitro translation of SP6 generated pEU RNA,the labeled products were immunoprecipitated with normal human serum(lane 1); HIV-1 infected human serum (lane 2); HIV-1 infected patientserum in the presence of 500 mM of peptide 2 (lane 3); HIV-2 infectedhuman serum (lane 4) or SIV-infected Rhesus macaques serum (lane 5). Theimmunoprecipitates were resolved on 15% SDS-PAGE. The ability of theanti-peptide 2 serum to recognize the 15 kD and 16 kD proteins wasexamined in three cell lines that express constitutively HIV-1 proteinsencoded by the 3' half of the virus. [These cell lines were prepared asdescribed in U.S. patent applications Ser. Nos. 806,263 and 865,151which are incorporated herein by reference.] Cloned HeLa cell lines thathave the region between the art gene and the 3' LTR of the proviral Eli,HXBc2 and Mal strains [Alizon, M. et al, Cell 46, supra] of HIV stablyintegrated were constructed and then isolated using standard techniques.The parental cells used to isolate these cell lines had previously beenselected for constitutive expression of the tat gene product, followinginfection with a retroviral vector carrying the tat coding sequences[Rosen, C.A., et al, J. Virol. 57: 379-384 (1985) which is incorporatedherein by reference]. Cells were labeled with [³⁵ S] methionine andcysteine and cell lysates were immunoprecipitated as described [Lee, T.H., et al, Proc. Natl. Acad. Sci, USA 81: 7579-7583 (1984)]. These celllines constitutively produce both the art and env gene products. Theplasmids used for construction of these cell lines contained the HIV LTRjuxtaposed 5' to the initiation codon of the art gene. The tat geneproduct was supplied in trans. FIG. 3A shows HeLa tat cell line lysatesimmunoprecipitated with anti-peptide 2 serum (lane 1) or HIV-1 infectedpatient serum (lane 2). FIG. 3B shows HeLa tat Eli lysatesimmunoprecipitated with preimmune rabbit serum (lane 1); antipeptide 2serum (lane 3); anti-peptide 2 serum in the presence of 500 μM ofpeptide 2 (lane 4); normal human serum (lane 5); HIV-1 infected patientserum (lane 6). Lane 2 represent an immunoprecipitation of labeled invitro translated product from pEU RNA with anti-peptide 2 serum. FIG. 3Cshows HeLa tat Mal lysate immunoprecipitated with preimmune rabbit serum(lane 1); anti-peptide 2 serum (lane 2); normal human serum (lane 3) andHIV-1 infected patient serum (lane 4). FIG. 3D shows HeLa tat III Blysate immunoprecipitated with preimmune rabbit serum (lane 1);anti-peptide 2 serum (lane 2) normal human serum (lane 3) and HIV-1infected patient serum (lane 4). The data of FIG. 3 demonstrates thatthe anti-peptide 2 antiserum specifically recognized a 15 kD protein inthe cell line derived from the Eli provirus (lane 3) that comigrateswith the 15 kD protein made in vitro (lane 2). The same antiserum doesnot recognize a protein in the cell line that expresses proteins derivedfrom the MAL (FIG. 3C) or the HXBc2 (FIG. 3D) proviruses. This is thepredicted result as neither the HXBc2 nor Mal proviruses contain aproperly positioned initiation codon (FIG. 2B). The absence of detectionof the 15 kD protein by the HIV-1 patient antiserum in the cell linederived from the Eli provirus is apparently due to both the low anti-vputiter in the patient antiserum used and the much smaller amount of the15 kD protein in the cell line compared to the in vitro translationproducts.

To examine the function of the U protein on the viral life cycle, weconstructed a recombinant provirus in which sequences in HXBc2 between aSalI site at position 5332 and a BamH1 site at position 8017 (+1 beingthe transcription initiation site) were replaced with the correspondingsequences from clone BH10.

Unlike HXBc2, BH10 possesses the AUG initiation codon for the U protein.BH10 is otherwise closely homologous to HXBc2. The net result of thisrecombination was to generate a proviral clone very similar to HXBc2except for a small number of conservative amino acid changes in the tat,art and env gene products, and the ability to utilize the U open readingframe. Transfection of Jurkat cells with HXBc2 and the recombinant BH10clones both resulted in production of virus, but spread of the BH10derived virus through the culture was significantly slower than thespread of the HXBc2-derived virus.

Aliquots of each culture were metabolically labelled 2, 4, and 7 dayspost-transfection with ³⁵ S-cystein and ³⁵ S-methionine. Extracts of thecells were then immunoprecipitated with AIDS-patient antiserum and runout on a 12.5% polyacrylamide gel. An autoradiogram of the gel is shownin FIG. 5B, Lanes 1, 4, and 7-control cells 2, 4, and 7 dayspost-transfection, respectively; lanes 2, 5, and 8-cells transfectedwith HXBc2, days 2, 4, and 7, respectively; lanes 3, 6, 9-cellstransfected with BH10 recombinant provirus, days 2, 4, and 7,respectively.

It is evident that those skilled in the art, given the benefit of theforegoing disclosure, may make numerous modifications thereof anddepartures from the specific embodiments described herein, withoutdeparting from the inventive concepts and the present invention is to belimited solely by the scope and spirit of the appended claims.

We claim:
 1. A DNA segment encoding an HIV-1 vpu gene product, whereinthe DNA segment does not encode an entire HIV env gene product.
 2. Avector containing:(a) the DNA segment of claim 1; and (b) a promoterupstream of the DNA segment.
 3. The vector of claim 2 wherein thepromoter is a viral promoter and the vector also contains an enhancerand polyadenylation sequences.
 4. A vector comprising:(a) a DNA segmentcomprising a sufficient number of nucleotides corresponding tonucleotides in region 5541-8017 of the HIV-1 genome, wherein an AUGcodon is inserted immediately upstream and in proper reading frame withsaid region to encode an HIV-1 vpu viral protein, wherein the DNAsegment does not encode an entire HIV env gene product; and (b) apromoter upstream of the DNA segment.
 5. The vector of claim 4, whereinthe sufficient number of nucleotides corresponds to the U open readingframe of the Eli strain of HIV-1.